Compositions and methods for reducing fucosylation of glycoproteins in insect cells and methods of use thereof for production of recombinant glycoproteins

ABSTRACT

Compositions for reducing fucosylation of glycoproteins in insect cells are provided. Also disclosed are methods of use of such compositions for the production of recombinant humanized proteins.

PRIORITY CLAIM

This application is a continuation application of U.S. patent application Ser. No. 14/504,195, filed Oct. 1, 2014, which claims priority to U.S. Provisional Application No. 61/885,294 filed Oct. 1, 2013. These applications are incorporated herein by reference as though set forth in full.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. GM049734 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and production of glycoproteins lacking fucosylation. More specifically, the invention provides compositions and methods for expressing a GDP-4-dehydro-6-deoxy-D-mannose reductase enzyme encoded by a recombinant baculovirus vector that blocks the production of GDP-L-fucose and generates a molecule lacking or having a reduced amount of fucose.

BACKGROUND OF THE INVENTION

Numerous publications and patent documents, including both published applications and issued patents, are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

In view of bioinformatic analyses suggesting that well over half of all human proteins are glycosylated, the ability to support glycosylation is an increasingly significant attribute of recombinant protein production systems, including insect-based systems, such as the baculovirus-insect cell system (reviewed by Jarvis 2009; Usami et al. 2010). The native human protein glycosylation process often results in the addition of terminally sialylated N-glycans that dramatically influence key properties, such as the in vivo half-lives, of glycoproteins in the human body (Ngantung et al. 2006; reviewed by Varki and Gagneux 2012). Most investigators know that insect-based systems, including the baculovirus-insect cell expression system, support recombinant protein glycosylation, but it is also important to recognize that these systems cannot produce human-type, terminally sialylated N-glycans (Marchal et al. 2001; Hillar and Jarvis 2010). This limitation has been addressed by glycoengineering baculovirus vectors and/or insect cell lines to encode and express mammalian glycogenes that extend endogenous insect cell N-glycan processing capabilities (Jarvis and Finn 1996; Wagner et al. 1996; Hollister et al. 1998; Ailor et al. 2000; Seo et al. 2001; Jarvis et al. 2001; Hollister and Jarvis 2001; Hollister et al. 2002; Aumiller et al. 2003; Tomiya et al. 2003; Chang et al. 2003; Yun et al. 2005; Hill et al. 2006; Okada et al. 2010; Aumiller et al. 2012; Geisler and Jarvis 2012; Palmberger et al. 2012; Mabashi-Asazuma et al. 2013). These efforts ultimately yielded new baculovirus-insect systems that can produce recombinant glycoproteins with human-type, terminally sialylated N-glycans. This was an important step towards the broader goal of extending the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production, which is not currently considered to be a legitimate application of this system. In order to finally achieve this goal, however, it also will be necessary to eliminate core α1,3-fucosylation of recombinant glycoproteins in certain insect cell lines, including some that are commonly used as hosts for baculovirus vectors, as this modification generates a highly immunogenic carbohydrate epitope (reviewed by Fotisch and Vieths 2001; Altmann et al. 2007).

There are two distinct types of N-glycan core fucosylation involving the addition of either α1,6- or α1,3-linked fucose residues to the core N-acetylglucosamine. Humans encode a single core fucosyltransferase, FUT8, which can only add α1,6-linked fucose residues, but many insects encode two core fucosyltransferases, FucT6 and FucTA, which can add α1,6- or α1,3-linked fucose residues, respectively, to the core N-acetylglucosamine (FIG. 1A; Fabini et al. 2001; Paschinger et al. 2005; Rendić et al. 2007). Because humans have no FucTA counterpart, we cannot produce core α1,3-fucosylated N-glycoproteins and, as a result, the core α1,3-fucosylated sugar epitope is immunogenic for many people. In fact, the presence of this epitope on bee venom glycoproteins accounts for the life-threatening allergic responses, such as anaphylactic shock, induced by bee stings in some humans (King et al. 1976; Prenner et al. 1992; Kubelka et al. 1995; reviewed by Altmann et al. 2007). In addition, pre-existing human antibodies against the immunogenic sugar epitope can give false positive results in diagnostic tests that use α1,3-fucosylated recombinant glycoproteins produced in the baculovirus-insect cell system (Hancock et al. 2008; Seismann et al. 2010).

Among the insect cell lines commonly used as hosts for baculovirus-mediated recombinant protein production, BTI-Tn-5Bl-4 (Wickham et al. 1992; commercialized as High Five™ by Life Technologies Inc., Carlsbad, Calif.), which is derived from Trichoplusia ni, produces high levels of the core α1,3-fucosylated N-glycan epitope, whereas Sf9 (Summers and Smith 1987), derived from Spodoptera frugiperda, and BmN (Maeda 1989), derived from Bombyx mori, do not (Rudd et al. 2000; Hancock et al. 2008; Seismann et al. 2010; Blank et al. 2011; Palmberger et al. 2011). Importantly, the capacity to produce immunogenic, core α1,3-fucosylated N-glycans trumps the opportunity to exploit the potentially higher recombinant glycoprotein production capacity of insect cell lines derived from Trichoplusia ni, such as High Five™ (Davis et al. 1992; Krammer et al. 2010).

While core α1,3-fucosylation is a relatively host-specific modification, core α1,6-fucosylation is a common feature of recombinant glycoproteins produced by all insect cell lines, including those used as hosts for baculovirus vectors. As noted above, core α1,6-fucosylation also occurs in humans and, therefore, does not produce an immunogenic sugar epitope. Nevertheless, this form of core fucosylation is also biotechnologically significant because it inhibits the effector functions of certain types of therapeutic antibodies (Shields et al. 2002; Shinkawa et al. 2003; reviewed by Satoh et al. 2006; Jefferis 2009), which comprise a large and growing share of the human biologics market (reviewed by Elvin et al. 2013). The inhibition of antibody effector functions by core fucosylation stimulated efforts to block pathways responsible for this modification and enable production of non-fucosylated antibodies in mammalian cell expression systems (FIG. 1B). Various approaches included repressing or eliminating FUT8 gene expression (Yamane-Ohnuki et al. 2004; Imai-Nishiya et al. 2007; Malphettes et al. 2010), overexpressing upstream processing enzymes to produce N-glycan structures that are not recognized as acceptor substrates (Ferrara et al. 2006; Zhong et al. 2012), and blocking production of GDP-L-fucose, which is required as the donor substrate for fucosylation by FUT8 (Imai-Nishiya et al. 2007; Kanda et al. 2007; von Horsten et al. 2010). To date, however, there have been no reported efforts to block recombinant glycoprotein fucosylation in any insect-based system, including the baculovirus-insect cell system, despite the arguably more serious problem of immunogenicity associated with core α1,3-fucosylation mediated by some insect and insect cell types.

SUMMARY OF THE INVENTION

In accordance with the present invention, a recombinant baculovirus expression vector for transient expression of GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) in an insect cell is disclosed. In one embodiment, the vector comprises the following operably linked components, i) an expression control sequence functional early in infection operably linked to a codon optimized RMD encoding nucleic acid; and ii) an insertion site suitable for insertion of one or more nucleic acids encoding at least one heterologous protein of interest. In a particularly preferred embodiment, the vector is AcRMD shown in FIG. 14. In another embodiment, the vector further comprises a nucleic acid sequence encoding at least one heterologous protein of interest operably linked to a promoter which is active later in infection, e.g., a p6.9 or polyhedrin promoter inserted at said insertion site.

In certain embodiments, the expression control sequence includes a promoter selected from the group consisting of baculovirus immediate early and delayed early promoters and an inducible promoter. In a preferred embodiment, the expression control sequence also includes an enhancer element.

The vectors of the invention have utility in the production of non-fucosylated therapeutic proteins. Such therapeutic proteins include, without limitation, an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, or a blood clotting factor. Cells useful in the methods disclosed herein include, for example, Sf9, Sf21, expresSF+®, Tn368, High Five®, Tni PRO®, Ea4, Ao38, BmN, S2, and S2R+ cells.

Also provided is a method for producing at least one molecule of interest (e.g., a therapeutic protein) lacking fucose, comprising providing insect cells and introducing a baculovirus comprising at least one nucleic acid molecule encoding a codon optimized enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, and at least one additional nucleic acid encoding a protein of interest driven by a promoter active later in infection that may or may not be on the same vector encoding RMD. Using this approach, inhibition of fucosylation is stabilized permitting production of a non-fucosylated protein of interest. The infected cells are incubated under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose. Following production, the non-fucosylated protein of interest is isolated.

In yet another aspect, a kit for the production of at least one protein of interest lacking fucose is provided comprising at least one recombinant baculovirus comprising at least one nucleic acid molecule encoding the codon optimized enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection, thereby stabilizing inhibition of fucosylation, and an insertion site for at least one additional nucleic acid molecule encoding at least one protein of interest, for production of non-fucosylated proteins of interest. The kit may also contain insect cells and at least one additional baculoviral vector comprising a promoter suitable to drive expression of the protein of interest and an insertion site for the at least one nucleic acid encoding the protein of interest.

In a further embodiment, the invention provides a method for production of a non-fucosylated protein in insect larvae. An exemplary method entails providing insect larvae and introducing therein a baculovirus comprising at least one nucleic acid molecule encoding a codon optimized enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an promoter active later in infection, thereby producing non-fucosylated proteins wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector. The infected larvae are then incubated under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose. The method further includes the step of isolating the protein of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Pathways of N-glycan fucosylation (FIG. 1A) and GDP-L-fucose biosynthesis (FIG. 1B). In theory, GDP-L-fucose biosynthesis in eukaryotic cells can be blocked by expression of bacterial RMD, which converts GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose. Abbreviations: FUT8, human core α1,6-fucosyltransferase 8; FucT6, insect core α1,6-fucosyltransferase; FucTA, insect core α1,3-fucosyltransferase; GMD, GDP-D-mannose 4,6-dehydratase; Fx protein, GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase; GFR, Golgi GDP-L-fucose transporter; Rmd, GDP-4-dehydro-6-deoxy-D-mannose reductase; FUK, fucokinase; FPGT, fucose-1-phosphate guanylyltransferase.

FIGS. 2A-2B. Core α1,3-fucosylation of endogenous insect cell glycoproteins. Total proteins in Sf9, High Five™, or Tni PRO™ cell lysates were resolved by SDS-PAGE in 12% acrylamide gels and stained with Coomassie Brilliant Blue (FIG. 2A) or transferred to a PVDF membrane and analyzed by western blotting with primary anti-HRP rabbit IgG and secondary α-rabbit IgG conjugated to alkaline phosphatase (FIG. 2B).

FIGS. 3A-3D. Phylogenetic trees of genes involved in GDP-L-fucose biosynthesis. Phylogenetic analysis of FUK (FIG. 3A), FPGT (FIG. 3B), GMD (FIG. 3C), and Fx protein (FIG. 3D) genes was performed using the Maximum Likelihood method with MEGA5 software (Tamura et al., 2011). The bars indicate the number of substitutions per site. Abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos taurus; Gg, Gallus gallus; Xl, Xenopus laevis; Dr, Danio rerio; Dp, Daphnia pulex; Ce, Caenorhabditis elegans; At, Arabidopsis thaliana.

FIGS. 4A-4B. Cell surface fucosylation. Sf9 and polyclonal SfRMD (FIG. 4A) or High Five™ and polyclonal TnRMD (FIG. 4B) cells were seeded into culture plates, and then stained with AAL, as described in Materials and methods. The cell surface staining patterns are shown alongside corresponding phase contrast micrographs, as indicated.

FIG. 5. Sf9, SfRMD 2B2, High Five™ and TnRMD 6A6 cells were infected with a baculovirus vector encoding a 6×HIS-tagged Fc domain of mouse IgG2a (mIgG2a-Fc) under the control of the baculovirus p6.9 promoter and the mIgG2a-Fc was affinity-purified from the extracellular fractions, as described in Materials and methods. Samples were then treated with peptide-N⁴-(N-acetyl-β-glucosaminyl) asparagine amidase (PNGase)-F or reaction buffer alone, resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with AAL to detect fucose or with anti-mouse IgG to detect the protein.

FIG. 6. Impact of AcP(+)IE1-RMD co-infection on mIgG2a-Fc fucosylation. Sf9 or Tni PRO™ cells were infected with Acp6.9-mIgG2a-Fc, a recombinant baculovirus encoding mIgG2a-Fc, or with equal doses of Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD, a recombinant baculovirus encoding Rmd. The cell-free media were harvested at 48 hours post-infection and used to affinity purify each mIgG2a-Fc preparation for lectin blotting analysis with AAL (specific for fucose) or western blotting analysis with anti-HRP (specific for core α1,3 fucose) or anti-mouse IgG with (+) or without (−) PNGase-F pre-treatment, as indicated.

FIGS. 7A-7D. N-glycan profiling of various mIgG2a-Fc preparations by MALDI-TOF MS. mIgG2a-Fc preparations were produced and purified from Sf9 and Tni PRO™ cells infected with Acp6.9-mIgG2a-Fc alone or Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD, as described in the legend to FIG. 6. PNGaseAr was used to remove the N-glycans, which were then recovered, permethylated, and analyzed by MALDI-TOF MS, as described in Materials and methods. mIgG2a-Fc from Sf9 cells infected with Acp6.9-mIgG2a-Fc alone (FIG. 7A), Sf9 cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (FIG. 7B), Tni PRO™ cells infected with Acp6.9-mIgG2a-Fc alone (FIG. 7C), or Tni PRO™ cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (FIG. 7D). All molecular ions were detected as [M+Na]⁺, assigned, and annotated using the standard cartoon symbolic representations.

FIG. 8. Genetic maps of parental baculoviral vector, baculovirus transfer plasmid, and AcRMD. The new baculovirus vector designated AcRMD was isolated by replacing the 5′ regions of the AcMNPV chiA and v-cath genes in BacPAK6 (Kitts and Possee 1993) baculoviral DNA with an expression cassette encoding EGFP and Rmd under the control of dual, back-to-back ie1 promoters separated by the AcMNPV hr5 enhancer, as described in Materials and methods.

FIG. 9. Genetic maps of recombinant baculoviruses encoding anti-CD20-IgG. The recombinant baculoviruses designated Ac-αCD20-IgG and Acp6.9-αCD20-IgG were isolated by using homologous recombination to replace the lacZ sequence in the parental baculovirus, AcGT (Toth et al, 2011), with expression cassettes encoding the anti-CD20-IgG heavy and light chains under the control of dual, back-to-back polyhedrin or p6.9 promoters separated by the second intron of the Drosophila melanogaster white gene, respectively. Similarly, the recombinant baculoviruses designated AcRMD-αCD20-IgG and AcRMDp6.9-αCD20-IgG were isolated by using homologous recombination to replace the lacZ sequence of the parental baculovirus, AcRMD (this study), with the same expression cassettes used to isolate Ac-αCD20-IgG and Acp6.9-αCD20-IgG.

FIG. 10. Expression and secretion of anti-CD20-IgG under the control of late or very late baculoviral promoters. Sf9 cells were infected with AcMNPV, Ac-CD20-IgG, Acp6.9-αCD20-IgG, AcRMD-αCD20-IgG, or AcRMDp6.9-αCD20-IgG. At 48 hours post-infection, the cell-free culture media were collected as the extracellular fraction, the cells were lysed, and the clarified supernatants were collected as the intracellular fraction. Proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-human IgG Fc-specific (α-HC) or anti-human IgG κ chain-specific antibodies (α-LC).

FIGS. 11A-11B. Analysis of purified anti-CD20-IgG preparations from Sf9 and Tni PRO™. The anti-CD20-IgG preparations produced by Sf9 or Tni PRO™ cells infected with Acp6.9-αCD20-IgG or AcRMDp6.9-αCD20-IgG were affinity purified using protein A, as described in Materials and methods, resolved by SDS-PAGE under reducing (FIG. 11A) or non-reducing (FIG. 11B) conditions, and stained with Coomassie Brilliant Blue.

FIGS. 12A-12C. Fucosylation of an anti-CD20-IgG (rituximab) produced using baculovirus or AcRMD baculovirus vectors. Sf9 and Tni PRO™ cells were infected with either Acp6.9-αCD20-IgG or AcRMDp6.9-αCD20-IgG, and the cell-free media were harvested and used to affinity purify anti-CD20-IgG at 48 hours post-infection. The results of western blotting assays with anti-human IgG Fc (FIG. 12A, α-HC) or anti-human IgG κ chain (FIG. 12B, α-LC) and lectin blotting assays with AAL (FIG. 12C) with (+) or without (−) PNGase-F pre-treatment are shown.

FIGS. 13A-13D. N-glycan profiling of various anti-CD20-IgG preparations by MALDI-TOF MS. Anti-CD20-IgG was isolated from Sf9 or Tni PRO™ cells infected with Acp6.9-αCD20-IgG or AcRMDp6.9-αCD20-IgG, as described in the legend of FIG. 12. The N-glycans were removed by PNGaseAr treatment, recovered, permethylated, and analyzed by MALDI-TOF MS, as described in Materials and methods. Anti-CD20-IgG from Sf9 cells infected with Acp6.9-αCD20-IgG (FIG. 13A), Sf9 cells infected with AcRMDp6.9-αCD20-IgG (FIG. 13B), Tni PRO™ cells infected with Acp6.9-αCD20-IgG (FIG. 13C), or Tni PRO™ cells infected with AcRMDp6.9-αCD20-IgG (FIG. 13D). All molecular ions were detected as [M+Na]⁺, assigned, and annotated using the standard cartoon symbolic representations.

FIGS. 14A-14E. A plasmid map (FIG. 14A) showing the major genetic elements and text files (FIGS. 14B to 14E) of the hr5 enhancer, AcMNPV ie1 promoter, codon-optimized RMD encoding sequence and the optional EGFP encoding sequences in the transfer plasmid pΔChi/Cath-EGFP/RMD, which was used to isolate AcRMD (SEQ ID NO: 1). In certain embodiments the EGFP encoding sequences are removed from SEQ ID NO: 1.

FIGS. 15A-15D. FIGS. 15A-15D show the pVL1393-polh-antiCD20-IgG sequence containing the AcMNPV polyhedrin promoter, anti-CD20-IgG heavy chain coding sequence, and anti-CD20-IgG light chain coding sequence (SEQ ID NO: 2), which was used to isolate a daughter of AcRMD encoding anti-CD20 under the control of the polyhedrin promoter.

FIGS. 16A-16D. FIGS. 16A-16D show text files of pVL1393-p6.9-antiCD20-IgG sequence containing the AcMNPV p6.9 promoter, anti-CD20-IgG heavy chain coding sequence, and anti-CD20-IgG light chain coding sequence (SEQ ID NO: 3), which was used to isolate a daughter of AcRMD encoding anti-CD20 under the control of the p6.9 promoter.

DETAILED DESCRIPTION OF THE INVENTION

The ability to glycosylate recombinant proteins is an important attribute of insect-based, including baculovirus-insect cell expression systems, but some insect cell lines produce recombinant glycoproteins with core α1,3-fucosylated N-glycans, which are highly immunogenic and render products unsuitable for human use. To address this problem, we exploited a bacterial enzyme, GDP-4-dehydro-6-deoxy-D-mannose reductase (Rmd), which consumes the precursor to GDP-L-fucose. We expected this enzyme to indirectly block glycoprotein fucosylation by blocking the production of GDP-L-fucose, which is required as the donor substrate for this process. Initially, we genetically transformed two different insect cell lines to constitutively express Rmd and successfully isolated subclones with fucosylation-negative phenotypes. Surprisingly, however, we found that the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that the prior art involving host cell engineering is ineffective in insect systems. Thus, we constructed a novel baculovirus vector designed to express Rmd immediately after infection and to facilitate the insertion of genes encoding any glycoprotein of interest for expression at a later time after infection. We used this vector to produce a daughter encoding a therapeutic anti-CD20-IgG (rituximab) and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a non-fucosylated form of this antibody. These results indicate that the novel Rmd⁺ baculoviral vector we produced can be used to solve the problem of immunogenic core α1,3-fucosylated N-glycan production associated with insect cell systems, including the baculovirus-insect cell system. These results extend the utility of such systems, which, when used in conjunction with existing glycoengineered insect cell lines, now include therapeutic glycoprotein production, in particular, production of recombinant antibodies lacking fucose with enhanced effector functions.

Definitions

A “cell line” refers to cells that can be cultured in the lab for an indefinite period and are useful for producing large amounts of a protein of interest.

As used herein, the term “insect” includes an insect in any stage of development, including first through fifth instar larvae. For the production of non-fucosylated polypeptides of interest, a large larva, such as a third, fourth, or fifth instar larva is preferred. It will be evident to a skilled worker which insect stage is suitable for a particular purpose, such as for direct production of a glycosylated polypeptide of interest, for storage or transport of an insect to a different location, for generation of progeny, for further genetic crosses, or the like.

With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ direction) in the naturally occurring genome of the organism from which it originates. For example, the “isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote. Isolated nucleic acids refers to those suitable for insertion in the recombinant baculoviral vectors described herein, e.g., RMD encoding nucleic acids and those encoding the desired protein of interest.

With respect to protein, the term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.

The term “protein of interest” is sometimes used herein. The term refers to any protein and includes, without limitation, therapeutic proteins, antibodies, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, or a blood clotting factor.

The term “promoter region or expression control sequence” refers to the transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the coding region, or within the coding region, or within introns. Such sequences regulate expression of a polypeptide coded for by a polynucleotide to which it is functionally (“operably”) linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the term expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, domains within promoters, upstream elements, enhancers, elements that confer tissue or cell specificity, response elements, ribosome binding sequences, transcriptional terminators, etc.

Suitable expression control sequences that can function in insect cells will be evident to the skilled worker. In some embodiments, the coding sequences described herein may be operably linked to an expression control sequence from the virus, itself, or to another suitable expression control sequence. Suitable baculovirus vectors include those based on Autographa californica NPV, Orgyia pseudotsugata NPV, Lymantria dispar NPV, Bombyx mori NPV, Rachoplusia ou NPV, Spodoptera exigua NPV, Heliothis zea NPV, Galleria mellonella NPV, Anagrapha falcifera nucleopolyhedrovirus (AfNPV), Trichoplusia ni singlenuclepolyhedrovirus (TnSNPV). As discussed above, baculovirus-based vectors have been generated (or can be generated without undue experimentation) that allow the cloning of large numbers of inserts, at any of a variety of cloning sites in the viral vector. Thus, more than one heterologous polypeptide may be introduced together into a transgenic insect cell or insect of the invention. The viral vector can be introduced into an insect cell or insect by known methods, such as by in vitro inoculation (insect cells) or injection or oral ingestion (insect larvae). Among the many suitable “strong” promoters that can be used are the baculovirus p10, polyhedrin (polh), p6.9, capsid, and v-cath promoters. Among the many suitable “weak” promoters are the baculovirus ie1, ie2, ie0, etl, 39K (aka pp31), and gp64 promoters. Other suitable constitutive promoters include insect actin hsp70, α1-tubulin, and ubiquitin gene promoters; RSV and MMTV promoters, copia promoter, gypsy promoter, and the cytomegalovirus IE promoter. If it is desired to increase the amount of gene expression from a weak promoter, enhancer elements, such as the baculovirus enhancer elements, hr5 or hr2, among others, may be used in conjunction with the promoter.

In some embodiments of the invention, as is discussed in more detail elsewhere herein, it is desirable that an expression control sequence is regulatable (e. g., comprises an inducible promoter and/or enhancer element). Suitable regulatable promoters include, e.g., hsp70 promoters, the Drosophila metallothionein promoter, an insect ecdysone-regulated promoter, the Saccharomyces cerevisiae Gal4/UAS system, and other well-known inducible promoter systems. A Tet-regulatable molecular switch may be used in conjunction with any constitutive promoter, such as those described elsewhere herein (e. g, in conjunction with the CMV-IE promoter, or baculovirus promoters). Another type of inducible promoter is a baculovirus delayed early, late or very late promoter that is only activated following infection by a baculovirus.

In some embodiments, the expression control sequence comprises a tissue- or organ-specific promoter. Many such expression control sequences will be evident to the skilled worker.

The term “vector” refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. A “baculovirus expression vector” is an expression vector consisting of a recombinant baculovirus that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.

The term “operably linked” means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

The phrase “consisting essentially of” when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO: For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

Methods for designing and preparing constructs suitable for generating recombinant baculovirus vectors for infection of insect cells or insects are known to one of ordinary skill in the art. For these methods, as well as other molecular biology procedures related to the invention, see, e. g., Sambrook et al. 1989; Wu et al. 1997; and Ausabel et al. 1994-1999.

In a preferred embodiment, the baculovirus replicates until the host insect cell is killed. The insect cell or insect lives long enough to produce large amounts of the non-fucosylated polypeptide of interest. In another embodiment, a baculovirus is used that is attenuated or non-permissive for the host. In this case, the host is not killed by replication of the baculovirus, itself (although the host may be damaged by expression of the heterologous protein of interest).

The following materials and methods are provided to facilitate the practice of the present invention.

Plasmid Constructions

The Pseudomonas aeruginosa Rmd (GenBank: AAG08839.1) coding sequence was optimized for Spodoptera frugiperda, synthesized by GeneArt® (Life Technologies, Gaithersburg, Md.), and cloned into pMK-T to produce a plasmid designated pKan-RMD.

pIE1-RMD, a plasmid designed to express Rmd under the control of the AcMNPV ie1 promoter, was constructed by subcloning the BamHI-NotI fragment of pKan-RMD into the BamHI and NotI sites of pIE1TV4 (Jarvis et al. 1996).

pAcP(+)IE1-RMD, a baculovirus transfer plasmid, was constructed by subcloning the BamHI-NotI fragment of pIE1-RMD into the BglII and NotI sites of pAcP(+)IE1TV3 (Jarvis et al. 1996).

pDIE1-EGFP/RMDΔBsu36I, a plasmid designed to express both EGFP and Rmd under the control of dual, back-to-back AcMNPV ie1 promoters, was constructed in four sequential steps. First, the bovine growth hormone (BGH) polyadenylation (poly A) signal was amplified by polymerase chain reaction (PCR) with pcDNA3.1 (Life Technologies) as the template and PmeI-BGHpolyA-Fw and SpeI-BGHpolyA-Rv as the primers. The resulting amplimer was digested with PmeI and SpeI and cloned into the corresponding sites of pIE1TV4 to produce a plasmid designated pIE1-BR. Second, the EGFP coding sequence was amplified by PCR with pIE1-EGFP as the template and BamHI/KpnI-EGFP-Fw and NruI-EGFPstop-Rv as the primers. The resulting amplimer was digested with BamHI and NruI and subcloned into the BamHI and PmeI sites of pIE1-BR to produce a plasmid designated pIE1-EGFP-BGH. Third, the Bsu36I site of Rmd was deleted by overlapping PCR mutagenesis. This involved amplifying the 5′-region of Rmd with NruI-RMD-Fw and RMD-BsuDel-Rv as the primers and the 3′-region of Rmd with RMD-BsuDel-Fw and ApaI-RMD-Rv as the primers, with pIE1-RMD as the template in both cases. The resulting 5′- and 3′-region Rmd amplimers were mixed and used as templates for an overlapping PCR with NruI-RMD-Fw and ApaI-RMD-Rv as the primers. The resulting RMDBsu36I amplimer was subcloned into pGEM-T (Promega, Madison, Wis.) to produce pDIE1-EGFP/RMDΔBsu36I. Finally, the EGFP-BGH poly A signal and RMDΔBsu36I coding sequence were assembled by sequentially subcloning the KpnI-HindIII fragment of pIE1-EGFP-BGH and the NruI-ApaI fragment of pGEMT-RMDΔBsu36I into the corresponding sites of pDIE1-TOPO.3 (Shi et al. 2007) to produce the plasmid designated pDIE1-EGFP/RMDΔBsu36I.

To construct a baculovirus transfer vector targeting the chiA/v-cath locus, we produced three PCR amplimers comprised of AcMNPV orf124/lef7, chiA, or v-cath/gp64 sequences and independently subcloned each one into pGEM-T. After verifying their sequences, these three DNA fragments were assembled by sequentially subcloning the PmeI-ApaI fragment of chiA and then the ApaI fragment of v-cath/gp64 into the corresponding sites of pGEMT-orf124/lef7 to produce a new plasmid designated pChi/CathTVC6. Finally, we blunt-ended the HindIII-BamHI fragment of pDIE1-EGFP/RMDΔBsu36I with T4 DNA polymerase (New England Biolabs, Beverly, Mass.) and inserted the resulting DNA fragment into the T4 DNA polymerase blunt-ended HindIII and EcoRI sites of pChi/CathTVC6. This transfer plasmid, which was designated pΔChi/Cath-EGFP/RMDΔBsu36I, was used to create the new baculovirus vector in which the viral chiA and v-cath genes were replaced by genes encoding Rmd and EGFP, each under the control of the AcMNPV ie1 promoter.

pVL1393-polh-αCD20-IgG and pVL1393-p6.9-αCD20-IgG, which are baculovirus transfer plasmids used to isolate baculovirus expression vectors encoding anti-CD20-IgG, were constructed in four sequential steps. First, the BGH poly A signal was PCR amplified with pIE1-BR as the template and EcoRI-BGHpolyA-Fw and EcoRV-BGHpolyA-Rv as the primers. The resulting amplimer was digested with EcoRI and EcoRV and then subcloned into the corresponding sites of pVL1393 (Summers and Smith 1987) to produce pVL1393-BGH. Second, the NotI-SbfI fragment of pGEMT-αCD20HC, which encodes the heavy chain, and the NotI-EcoRI fragment of pGEMT-αCD20LC, which encodes the light chain of anti-CD20-IgG were sequentially subcloned into the NotI-PstI and NotI-EcoRI sites of pVL1393-BGH to produce pVL1393-αCD20-IgG-no promoter. Third, two copies of the AcMNPV polyhedrin or p6.9 promoter were assembled in back-to-back orientation, with the second intron of the Drosophila melanogaster white gene as an intervening spacer, to create plasmids designated either pGEMT-ppol-wi-ppol or pGEMT-p6.9-wi-p6.9, respectively. The former was constructed by PCR-amplifying two copies of the polyhedrin promoter with pAcGT N-term 8×His pPol (Toth et al. 2011) as the template and either NheI-ppol-Fw and NotI-ppol-Rv or XhoI-ppol-Fw and SphI/PmeI-ppol-Rv as the primers. The resulting amplimers were digested with NheI and NotI or XhoI and SphI, respectively, and sequentially subcloned into the corresponding sites of pGEM-WIZ (Bao and Cagan 2006). Similarly, pGEMT-p6.9-wi-p6.9 was constructed by PCR-amplifying two copies of the p6.9 promoter with pAcp6.9GT N-term 8×His pPol (Toth et al. 2011) as the template and either NheI-p6.9-Fw and NotI-p6.9-Rv or XhoI-p6.9-Fw and SphI/PmeI-p6.9-Rv as the primers. The resulting amplimers were digested with NheI and NotI or XhoI and SphI, respectively, and sequentially subcloned into the corresponding sites of pGEM-WIZ. Finally, each back-to-back promoter cassette was independently subcloned into the anti-CD20-IgG baculovirus transfer plasmid described above. pVL1393-polh-αCD20-IgG was constructed by subcloning the PmeI-NotI fragment of pGEMT-ppol-wi-ppol and pVL1393-p6.9-αCD20-IgG was constructed by subcloning the PmeI-NotI fragment of pGEMT-p6.9-wi-p6.9 into the corresponding sites of pVL1393-αCD20-IgG_no promoter, respectively.

Phusion® Taq DNA polymerase (New England Biolabs) and a Biometra TProfessional Standard Thermocycler (Gottingen, Germany) were used for all PCRs and all primer sequences are given in Table 1.

TABLE 1 Primers used in this study. Primer name Sequence (5′ to 3′) PmeI-BGHpolyA-Fw Gtttaaacgcctcgactgtgccttctagttg  (SEQ ID NO: 4) SpeI-BGHpolyA-Rv Actagttccccagcatgcctgctatt  (SEQ ID NO: 5) BamHI/KpnI-EGFP-Fw Aatggatccggtaccaccatggtgagcaagggcg  (SEQ ID NO: 6) NruI-EGFPstop-Rv Ggcacttcgcgattacttgtacagctcgtccatgcc  (SEQ ID NO: 7) NruI-RMD-Fw Ttatctcgcgaaccatgactcaacgcttgttcg  (SEQ ID NO: 8) RMD-BsuDel-Rv Atcacggaatgcctcgggtacgtatg  (SEQ ID NO: 9) RMD-BsuDel-Fw Gctggtcaaacatacgtacccgaggc  (SEQ ID NO: 10) ApaI-RMD-Rv Tgggcccttactcctctctaacacgagattccca  (SEQ ID NO: 11) EcoRI-BGHpolyA-Fw Attgtgaattcgcctcgactgtgccttctagttgc  (SEQ ID NO: 12) EcoRV-BGHpolyA-Rv Taattgatatctccccagcatgcctgctattg  (SEQ ID NO: 13) NheI-ppol-Fw Gccgcgctagcatcatggagataattaaaatgataaccatctcgcaaataa  (SEQ ID NO: 14) NotI-ppol-Rv Aatttgcggccgcagcgcccgatggtgggacg  (SEQ ID NO: 15) XhoI-ppol-Fw Gggcctcgagatcatggagataattaaaatgataaccatctcgcaaataa  (SEQ ID NO: 16) SphI/PmeI-ppol-Rv Atttgcatgcgtttaaacagcgcccgatggtgggacg  (SEQ ID NO: 17) NheI-p6.9-Fw Gccgcgctagcaaattccgttttgcgacgatg  (SEQ ID NO: 18) NotI-p6.9-Rv Aatttgcggccgcgtttaaattgtgtaatttatgtagctgtaatttttacc  (SEQ ID NO: 19) XhoI-p6.9-Fw Gggcctcgagaaattccgttttgcgacgatg  (SEQ ID NO: 20) SphI/PmeI-p6.9-Rv Atttgcatgcgtttaaacgtttaaattgtgtaatttatgtagctgtaatttttacc  (SEQ ID NO: 21) orf124-Fw Atttgtatttaatcaatcgaaccgtgcac  (SEQ ID NO: 22) RMD-Rv Gattgggaatctcgtgttagagaggagtaa  (SEQ ID NO: 23) EGFP-Fw Atcttcttcaaggacgacggcaac  (SEQ ID NO: 24) gp64-Rv Agcaagatggtaagcgctattgttttatatgtgc  (SEQ ID NO: 25) chi-Fw Agatgggtatgaaaccatacaacaagtgtg  (SEQ ID NO: 26) cath-Rv Cgctaccataatctttgttgaatcgatg  (SEQ ID NO: 27) Cells and Viruses

Sf9 and Tni PRO™ (Expression Systems, Woodland, Calif.) cells were routinely maintained as shake-flask cultures in ESF 921 medium (Expression Systems) at 28° C. High Five™ cells (Life Technologies) were maintained as adherent cultures in TNM-FH medium containing 10% (v/v) fetal bovine serum (Atlanta Biologics, Atlanta, Ga.).

SfRMD is a transgenic Sf9 cell derivative that was produced for this study using a modification of an established procedure (Harrison and Jarvis, 2007). Briefly, Sf9 cells were co-transfected with a mixture of pIE1-Neo (Jarvis et al. 1990) and pIE1-RMD using a modified calcium phosphate method (Summers and Smith 1987). The transfected cells were allowed to recover for 1 day, treated with 1 mg of G418 (Life Technologies)/mL of TNM-FH medium containing 10% (v/v) fetal bovine serum (Atlanta Biologics) for 1 week, and G418-resistant clones were isolated by limiting dilution, as described previously (Hollister and Jarvis 2001). After stepwise amplification into larger cultures, individual clones were assayed by cell surface staining with a fucose-specific lectin, as described below. Unstained clones were designated SfRMD, characterized in more detail, and used for downstream experiments, as described below.

TnRMD is a transgenic High Five™ cell derivative that was designed to constitutively express the Rmd gene and produced for this study as described above for SfRMD. The baculovirus expression vector designated AcmIgG2a-Fc has been described previously (Geisler and Jarvis 2012).

The baculovirus expression vector designated AcP(+)IE1-RMD was isolated by co-transfecting Sf9 cells with a mixture of pAcP(+)IE1-RMD and BacPAK6 (Kitts and Possee 1993) baculoviral DNA after pre-linearizing the latter by digestion with Bsu36I. Viral progeny were harvested, clones were resolved by plaque assay in the presence of X-Gal (Sigma-Aldrich; St. Louis, Mo.), and an isolated clone with a white plaque phenotype was amplified and titered in Sf9 cells.

Baculovirus expression vectors designated Ac-αCD20-IgG and Acp6.9-αCD20-IgG were isolated by co-transfecting Sf9 cells with mixtures of either pVL1393-polh-αCD20-IgG or pVL1393-p6.9-αCD20-IgG and AcGT baculoviral DNA (Toth et al. 2011) after pre-linearizing the latter by digestion with Bsu36I. The transfected cells were cultured for 5 days in growth medium containing 100 μM ganciclovir (Life Technologies) and then viral progeny were harvested, clones were resolved by plaque assay, and an isolated clone with a white plaque phenotype was amplified and titered in Sf9 cells, as described above.

The baculovirus expression vector designated AcRMD was isolated by co-transfecting Sf9 cells with a mixture of pΔChi/Cath-EGFP/RMDΔBsu36I and BacPAK6 (Kitts and Possee 1993) baculoviral DNA (FIG. 14). This recombination strategy was custom-designed to replace the AcMNPV chiA and v-cath coding sequences with Rmd and EGFP coding sequences placed under the control of dual AcMNPV ie1 promoters separated by the AcMNPV hr5 enhancer. Viral progeny were harvested, clones were resolved by plaque assay, and fluorescent plaques were identified using an Axiovert 25 microscope equipped with HBO 50 epi-fluorescence unit (Carl Zeiss, Oberkochen, Germany). Several plaques were picked, screened by PCR, as described below, and those that included viral progeny with the Rmd and EGFP genes in the correct genomic location were used for a second round of plaque purification. The PCR screen was repeated, promising clones were used for a third round of plaque purification, and well-isolated clones with fluorescent plaque phenotypes were picked and amplified in Sf9 cells. A Wizard genomic DNA purification kit (Promega) was used to extract total DNA from Sf9 cells infected with each of those viral clones and used as the templates for final PCR analyses, which were the same as those performed for the prior screening steps. In each case, we performed PCRs with two different primer pairs, orf124-Fw and RMD-Rv or EGFP-Fw and gp64-Rv to determine if the final AcRMD isolates had the EGFP/RMDΔBsu36I expression cassette in the correct genomic location, that is, in place of the viral chiA and v-cath genes. In addition, we used a third primer pair, chi-Fw and cath-Rv, to directly confirm the absence of those viral genes and show that the final AcRMD isolates were not detectably contaminated with parental BacPAK6 vector DNA. Three clones were positive with the first two primer pairs and negative with the third and one clone was designated AcRMD, further amplified in Sf9 cells, and used for the remainder of this study.

Baculovirus expression vectors designated AcRMD-αCD20-IgG and AcRMDp6.9-αCD20-IgG (FIGS. 15 and 16) were isolated by co-transfecting Sf9 cells with a mixture of pVL1393-polh-αCD20-IgG or pVL1393-p6.9-αCD20-IgG and AcRMD baculoviral DNA pre-linearized by digestion with Bsu36I. Viral progeny were harvested, clones were resolved by plaque assay, and an isolated clone with a white plaque phenotype was amplified and titered in Sf9 cells, as described above.

Cell Surface Lectin Staining

Sf9, SfRMD, High Five™, or TnRMD cells were stained with a mixture of biotinylated AAL (Vector Labs, Burlingame, Calif.) and fluorescein-conjugated streptavidin (Vector Labs) in lectin staining buffer (10 mM Hepes, pH 7.5, 50 mM NaCl, 1 mM CaCal₂, 1 mM MgCl₂, 1 mM MnCl₂) for 10 min at room temperature. The cells were then washed twice with lectin staining buffer and imaged using an Olympus FSX 100 fluorescence microscope (Tokyo, Japan).

Recombinant Protein Expression and Purification

For small scale recombinant protein expression experiments, Sf9 cells were seeded at a density of 2×10⁶ cells/well into 6-well plates and infected with Ac-αCD20-IgG, Acp6.9-αCD20-IgG, AcRMD-αCD20-IgG, or AcRMDp6.9-αCD20-IgG at a multiplicity of about 2 plaque forming units/cell. The virus was allowed to adsorb for 1 hour and then the infected cells were washed once with 1 mL of ESF 921 and cultured in 1 mL of fresh ESF 921 at 28° C. to 48 hours post-infection. At that time, the cell-free media were prepared by low speed centrifugation and used as the extracellular fractions for downstream analysis. The cell pellets were washed once with phosphate buffered saline, lysed with 1 mL of extraction buffer (20 mM HEPES, pH 7.4, 0.15 M NaCl, 0.1 mM EDTA, 0.5% Nonidet P-40), clarified by centrifugation at 13,000 rpm for 10 min, and the supernatants were used as the intracellular fractions for downstream analysis.

For larger scale recombinant protein expression and purification, Sf9, Tni PRO™, or High Five™ cells were seeded into 50 mL shake flask cultures at a density of 2×10⁶ cells/mL in ESF 921 and then infected with Acp6.9-αCD20-IgG, AcRMDp6.9-αCD20-IgG, AcmIgG2a-Fc, or a mixture of AcmIgG2a-Fc and AcP(+)IE1-RMD using a multiplicity of about 2 plaque forming units/cell for each virus. After a 1 hour adsorption period, the infected cells were gently pelleted, resuspended in 50 mL of fresh ESF 921 supplemented with antibiotics (1.25 μg/mL amphotericin B and 25 μg/mL gentamicin), returned to the shake flasks, and incubated at 28° C. to 48 hours post-infection. Cells and debris were pelleted by centrifugation at 1,000×g for 10 min at 4° C., the supernatants were harvested, and budded baculovirus progeny were removed by centrifugation at 70,000×g for 30 min at 4° C. One Complete Protease Inhibitor Cocktail tablet (Roche Diagnostics, Indianapolis, Ind.) was dissolved in each final supernatant and then each was transferred into a 12-14,000 molecular weight cut-off membrane (Spectrum Labs, Rancho-Dominguez, Calif.) and dialyzed against 0.1 M NaCl for 6 hours. Each anti-CD20-IgG or mIgG2a-Fc preparation was subsequently dialyzed against 0.15 M or 0.5 M NaCl, respectively, and then each was purified by was affinity-purified Protein A agarose (GenScript, Piscataway, N.J.) or ProBond nickel (Life Technologies) affinity chromatography, respectively, according to the manufacturer's instructions. Eluted fractions containing the purified proteins were pooled and desalted on PD10 columns (GE Healthcare) equilibrated with phosphate buffered saline.

SDS-PAGE, Western Blotting, and Lectin Blotting Analyses

Several different types of samples were isolated from Sf9, Tni PRO™, and/or High Five™ cells and used for SDS-PAGE, western blotting, and/or lectin blotting analysis. These included intracellular fractions from uninfected cells, extracellular fractions from cells infected with various recombinant baculoviruses, and anti-CD20-IgG or mIgG2a-Fc purified from cells infected with various recombinant baculoviruses, as described above. In some cases, the samples were pre-treated with PNGase-F in reaction buffer (New England Biolabs) or PNGase-F reaction buffer alone according to the manufacturer's instructions, as described in the Figure legends. All samples were boiled in Laemmli sample buffer and then proteins were resolved by SDS-PAGE on 12% polyacrylamide gels and either stained with Coomassie Brilliant blue, destained, and imaged or electrophoretically transferred to Immobilon-P membranes (Millipore). The latter were blocked for 1 hour at room temperature with Tris-buffered saline (150 mM NaCl in 50 mM Tris-HCl, pH 7.5) containing either 5% bovine serum albumin (w/v; Sigma-Aldrich) and 0.5% (v/v) Tween 20 (Sigma-Aldrich) or 1% Tween 20 for western or lectin blotting assays, respectively. After blocking, western blotting assays were completed using alkaline phosphatase-conjugated goat anti-human IgG κ chain (Sigma-Aldrich) to detect the anti-CD20-IgG light chain, alkaline phosphatase-conjugated goat anti-human IgG Fc (Sigma-Aldrich) to detect the anti-CD20-IgG heavy chain, alkaline phosphatase-conjugated goat anti-mouse IgG to detect mIgG2a-Fc, or rabbit anti-HRP IgG (Gentaur, Brussels, Belgium) as the primary and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich) as the secondary antibody to detect core α1,3-liked fucose. The lectin blotting assays were completed using alkaline phosphatase-conjugated AAL (Vector Laboratories) and the probes used for all western and lectin blotting assays were detected using a standard chromogenic assay for alkaline phosphatase activity (Blake et al. 1984).

Mass Spectrometry

N-glycans were enzymatically released from various purified mIgG2a-Fc and anti-CD20-IgG preparations by exhaustive digestion with PNGaseAr (New England Biolabs). The spent reactions were applied to pre-conditioned C18 SepPak cartridges (Waters Corp., Milford, Mass.) and the flow-through and a 5% (v/v) aqueous acetic acid wash were pooled, evaporated, and permethylated, as described previously (Dell et al. 1994). The permethylated N-glycan derivatives were extracted into chloroform, pooled with several aqueous washes, re-evaporated, and then resuspended in acetonitrile, mixed 1:1 with 2,5-dihydroxybenzoic acid matrix (10 mg/mL in 50% aqueous acetonitrile), and samples were spotted onto the MALDI-TOF target plate. Data acquisition was performed manually on a Model 4700 Proteomics Analyzer equipped with an Nd:YAG laser (Applied Biosystems, Framingham, Mass.) and 1,000 shots were accumulated in the reflectron positive ion mode.

The following example is provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way.

Example I A Novel Baculovirus Vector for the Production of Non-Fucosylated Recombinant Glycoproteins in Insect Cells Analysis of Core α1,3-Fucosylation in Three Insect Cell Lines

High Five™ cells, derived from Trichoplusia ni, but not Sf9 cells, derived from Spodoptera frugiperda, produce core α1,3-fucosylated glycoproteins (Rudd et al. 2000; Seismann et al. 2010; Blank et al. 2011; Palmberger et al. 2011). Another Trichoplusia ni cell line used as a host for baculovirus expression vectors is Tni PRO™ (Kwon et al. 2009; Bourhis et al. 2010; Bongiovanni et al. 2012; He et al. 2013; Merchant et al. 2013), but its capacity for core α1,3-fucosylation has not been reported. Thus, we analyzed intracellular extracts of uninfected Tni PRO™ cells by western blotting with anti-horseradish peroxidase (HRP), which detects core α1,3-linked fucosylation, using extracts from Sf9 and High Five™ cells as negative and positive controls. Coomassie brilliant blue staining showed that approximately equal amounts of protein were loaded in each case (FIG. 2A). The anti-HRP antibody did not detectably react with the Sf9 lysates, but reacted with several glycoproteins in the High Five™ lysates, as expected (FIG. 2B). In addition, this antibody reacted with several glycoproteins in the Tni PRO™ lysates (FIG. 2B), indicating that Tni PRO™ cells produce the immunogenic core α1,3-fucosylated sugar epitope at levels roughly comparable to High Five™ cells. These results show that it will be necessary to block core α1,3-fucosylation in both of these cell lines before we can exploit their potentially higher capacity for recombinant glycoprotein production (Davis et al. 1992; Krammer et al. 2010).

Glycoengineering Insect Cells to Block Glycoprotein Fucosylation

Our plan to block glycoprotein fucosylation in insect cell lines focused on blocking the biosynthesis of GDP-L-fucose, which is the donor substrate required for this process. This was a particularly attractive approach in our system because insects appeared to be the only multicellular organisms lacking two enzymes, FUK and FPGT, required for the GDP-L-fucose salvage pathway in other organisms (FIG. 1B). We drew this conclusion from a previous study indicating there are no FUK and FPGT orthologs in the Drosophila melanogaster genome, which was the only insect genome sequenced at that time (Rhomberg et al. 2006). However, because we now have more information from silkworm, honeybee, and mosquito genome sequencing projects, among others, we also searched the NCBI database using mammalian FUK and/or FPGT genes as queries. We identified putative orthologs in some invertebrates, including arthropods and nematodes, but none in any insects (FIG. 3A-3B). In contrast, using genes required for de novo GDP-L-fucose synthesis as queries, we found putative orthologs in a wide variety of insects, as expected (FIG. 3C-3D). Although we could not exclude the possibility that insects have an unknown salvage pathway, these results strengthened the idea that we could effectively block GDP-L-fucose biosynthesis by blocking the de novo biosynthetic pathway, alone, in insect cell lines.

In principle, we might have achieved this goal by inactivating any of the genes encoding enzymes involved in this pathway, including GMD, Fx, GFR, or FUT8 (FIG. 1B). However, there are no reported examples of targeted gene knockouts in any lepidopteran insect cell line and this approach is technically complicated by the fact that neither the Spodoptera frugiperda nor the Trichoplusia ni genomes have been sequenced. On the other hand, we have reported many examples of foreign gene knock-ins using both Sf9 (Hollister et al. 1998; Hollister and Jarvis 2001; Hollister et al. 2002; Aumiller et al. 2003; Aumiller et al. 2012; Geisler and Jarvis 2012; Mabashi-Asazuma et al. 2013) and High Five™ (Breitbach and Jarvis 2001) cells, as part of our broader effort to glycoengineer the baculovirus-insect cell system. Thus, we pursued an analogous glycoengineering strategy that involved transforming Sf9 and High Five™ cells with a constitutively expressible Pseudomonas aeruginosa Rmd gene. This gene encodes an enzyme that consumes the immediate precursor to GDP-L-fucose to produce GDP-D-rhamnose, which we believed would be a dead-end product in insect cells (FIG. 1B; Rocchetta et al. 1998). Thus, we expected Rmd to block the production of GDP-L-fucose and glycoprotein fucosylation because GDP-L-fucose is required as the donor substrate for this process.

We constructed an expression plasmid encoding Rmd under the transcriptional control of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) immediate early 1 (ie1) promoter, as described in Materials and methods. We then co-transfected Sf9 and High Five™ cells with a mixture of this plasmid and an antibiotic-resistance marker and selected polyclonal transformed Sf9 and High Five™ cell subpopulations, which were designated SfRMD and TnRMD, respectively. We assayed both polyclonal transformed cell populations for cell surface fucosylation by staining with Aleuria aurantia lectin (AAL), as described in Materials and methods. The results showed that 100% of the parental Sf9 and High Five™ cells were stained, while only 23% of the SfRMD and 33% of the TnRMD cells were stained with AAL (FIG. 4). These results demonstrated that Rmd overexpression significantly reduced glycoprotein fucosylation in insect cells. We subsequently isolated single cell clones from the polyclonal transformed SfRMD and TnRMD populations, identified several that exhibited no detectable AAL cell surface staining, and amplified one of each, designated SfRMD 2B2 and TnRMD 6A6. We then infected Sf9, SfRMD 2B2, High Five™ and TnRMD 6A6 cells with a baculovirus vector encoding a 6×HIS-tagged Fc domain of mouse IgG2a (mIgG2a-Fc) under the control of the baculovirus p6.9 promoter, which is activated during the late phase of infection (Passarelli and Guarino 2007), and affinity-purified the mIgG2a-Fc from the extracellular fractions, as described in Materials and methods. Samples were then treated with peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase (PNGase)-F or reaction buffer alone, resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with AAL to detect fucose or with anti-mouse IgG to detect the protein (FIG. 5).

The results of this analysis showed that the mIgG2a-Fc preparations produced by the parental cell lines were AAL-reactive and either completely (Sf9) or partially (High Five™) sensitive to PNGase-F, indicating they were core α1,6- (Sf9) or α1,6- and α1,3- (High Five™) fucosylated, respectively. In contrast, neither of the mIgG2aFc preparations from SfRMD 2B2 or TnRMD 6A6 cells was AAL-reactive, indicating that RMD effectively blocked glycoprotein fucosylation in both cell types. However, we were surprised to find that both of these prototype SfRMD and TnRMD clones recovered strong AAL reactivity after 38 and 35 passages in culture, respectively, indicating that the fucosylation-negative phenotype is unstable in these cells (data not shown). We obtained this same result with other SfRMD and TnRMD clones, which was extremely surprising, because this knock-in approach had been used previously to isolate a stable, fucosylation deficient CHO cell derivative (von Horsten et al. 2010) and because we had previously demonstrated that a transgenic insect cell line transformed with six different transgenes was stable for at least 300 passages in culture (Aumiller et al. 2012). Although we found that they still expressed the Rmd gene at the transcriptional level, we did not expend any further effort to determine how the SfRMD and TnRMD cells recovered their original fucosylation-positive phenotypes. Rather, after finding, much to our surprise, that our insect cell glycoengineering approach was unsuccessful, we began to develop a new approach for blocking glycoprotein fucosylation in the baculovirus-insect cell system.

Assessing a Vector-Based Glycoengineering Strategy for Blocking Glycoprotein Fucosylation

In view of the surprising phenotypic instability of SfRMD and TnRMD cells, we abandoned our efforts to glycoengineer the host cell component and focused our attention on the baculoviral vector component of the baculovirus-insect cell system. The baculovirus-insect cell system is a transient expression system in which baculovirus-infected insect cells express the glycoprotein of interest for a period of about 2 days, beginning about a day after infection, when the gene encoding the glycoprotein of interest begins to be expressed during the late or very late phase of infection. Baculovirus-infected cells are typically harvested by about 60-72 hours post-infection because the host cells die and lyse at later times of infection. Given the transient nature of this expression system, we realized that the phenotypic instability observed with long-term constitutive expression of Rmd in the host cells could be overcome by engineering the viral vector to express this enzyme. However, we also realized we would have to design the new vector to express Rmd early in infection, in order to reduce endogenous GDP-L-fucose to low enough levels to block fucosylation before the glycoprotein of interest began to be expressed during a later phase of infection.

To assess our ability to meet these requirements, we isolated a recombinant baculovirus designated AcP(+)IE1-RMD, which encodes Rmd under the control of the AcMNPV ie1 promoter and was expected to express the Rmd gene immediately after baculovirus infection (Guarino and Summers 1986). We then used this virus to examine the impact of immediate early Rmd expression on fucosylation of mIgG2a-Fc. This was accomplished by co-infecting Sf9 or Tni PRO™ cells with equal doses of AcP(+)IE1-RMD and Acp6.9-mIgG2a-Fc, or with Acp6.9-mIgG2a-Fc alone as a control, and then affinity-purifying the mIgG2a-Fc from the extracellular fractions for analysis, as described in Materials and methods. Samples of the purified mIgG2a-Fc were then treated with PNGase-F or reaction buffer alone, resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with AAL to detect fucose, anti-HRP to detect core α1,3-linked fucose, or anti-mouse IgG to detect the protein. The results showed that the control mIgG2a-Fc preparation produced by Sf9 cells infected with Acp6.9-mIgG2a-Fc alone were AAL-reactive (FIG. 6, top panel), indicating it was fucosylated. In addition, PNGase-F pre-treatment eliminated its AAL reactivity (FIG. 6, top panel), indicating that it was exclusively core α1,6-fucosylated, because PNGase-F does not remove core α1,3-fucosylated N-glycans (Tretter et al. 1991). This interpretation was supported by the fact that anti-HRP, which recognizes the immunogenic sugar epitope produced by core α1,3-fucosylation, did not react with this mIgG2a-Fc preparation (FIG. 6, middle panel). In contrast, the mIgG2a-Fc preparation from Sf9 cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD was not AAL-reactive (FIG. 6, upper panel), indicating it was not fucosylated and, therefore, that baculovirus-mediated Rmd expression blocked core α1,6-fucosylation in Sf9 cells. The mIgG2a-Fc from Tni PRO™ cells infected with Acp6.9-mIgG2a-Fc alone reacted not only with AAL (FIG. 6, top panel), but also with anti-HRP (FIG. 6, middle panel), whether or not it was pre-treated with PNGase-F, indicating that it was core α1,3-fucosylated. This interpretation was supported by the fact that PNGase-F did not detectably alter the electrophoretic mobility of this mIgG2a-Fc preparation (FIG. 6, bottom panel). In contrast, the mIgG2a-Fc from Tni PRO™ cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD had no detectable AAL (FIG. 6, top panel) or anti-HRP (FIG. 6, middle panel) reactivity and was sensitive to PNGase-F (FIG. 6, bottom panel). These results indicated that baculovirus-mediated Rmd expression blocked both core α1,6- and core α1,3-fucosylation in Tni PRO™ cells.

To more directly assess the impact of AcP(+)IE1-RMD co-infection on core fucosylation of mIgG2a-Fc, we enzymatically released, permethylated, and analyzed the N-glycans from each mIgG2a-Fc preparation in FIG. 6 by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), as described in Materials and methods. The results indicated that the major N-glycans isolated from the mIgG2a-Fc produced by Sf9 cells infected with Acp6.9-mIgG2a-Fc alone were mono-fucosylated (m/z 1345.7 and 1590.8; FIG. 7A). Based on the results shown in FIG. 6, these are most likely core α1,6-fucosylated N-glycans. In contrast, none of the peaks assigned as fucosylated N-glycans were detected in the mIgG2a-Fc produced by Sf9 cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (FIG. 7B). The major N-glycans isolated from the mIgG2a-Fc produced by Tni PRO™ cells infected with Acp6.9-mIgG2a-Fc alone included mono-, but predominantly di-fucosylated structures (FIG. 7C). While their masses cannot directly reveal the nature of the linkages between the fucose and core N-acetylglucosamine residues in these N-glycans, it is most reasonable to conclude that the di-fucosylated N-glycans have both α1,6- and α1,3-linked fucose residues. Again, none of the peaks assigned as mono- or di-fucosylated N-glycans were detected in the mIgG2a-Fc produced by Tni PRO™ cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (FIG. 7D). Together, the AAL blotting, anti-HRP antibody blotting, and MALDI-TOF MS data clearly demonstrate that baculovirus-mediated expression of Rmd at an early stage of infection can effectively block core α1,6- and α1,3-fucosylation of recombinant glycoproteins expressed later in infection in the baculovirus-insect cell system.

A Novel Baculovirus Vector Designed to Produce Non-Fucosylated Recombinant Glycoproteins

While co-infection with multiple baculoviruses is an approach that can be used to co-express two or more different recombinant proteins in insect cells, balanced co-infections can be difficult to achieve, infection with multiple baculoviruses can dramatically reduce recombinant glycoprotein yields, and optimal co-infection conditions must be established for each experiment (Sokolenko et al. 2012). We chose to circumvent these issues by isolating a novel baculovirus vector designed not only to express Rmd immediately after infection, but also to facilitate downstream insertion of genes encoding any glycoprotein of interest for expression at a later time of infection. The experimental design used to isolate this new baculovirus vector, designated AcRMD, included using the ie1-Rmd gene to replace portions of the viral chiA and v-cath genes, which encode a chitinase purported to interfere with secretory pathway function (Hawtin et al. 1997; Kaba et al. 2004; Hitchman et al. 2010) and a cathepsin-like protease that can degrade recombinant proteins of interest (Slack et al. 1995; Kaba et al. 2004; Hitchman et al. 2010), respectively. The complete details of the strategy used to isolate this new vector are given in Materials and methods and genetic maps of the parental viral genome, transfer plasmid, and final baculoviral vector are shown in FIG. 8 and the plasmid map shown in FIG. 14.

AcRMD Produces Non-Fucosylated Recombinant Glycoproteins

To examine the capabilities of the new baculovirus vector described in the preceding section, we isolated two independent daughter recombinants encoding rituximab, an anti-CD20-IgG, with the light and heavy chain genes placed under the independent transcriptional control of dual back-to-back promoters from either the AcMNPV p6.9 or polyhedrin genes (see genetic maps in FIG. 9). We chose rituximab as the model glycoprotein for this work because it is a biotechnologically relevant therapeutic antibody that is core α1,6-fucosylated in mammalian cells, has enhanced effector function in the absence of fucosylation, and has been used to treat rheumatoid arthritis and non-Hodgkin's lymphoma in human patients (Li et al. 2013; reviewed by Lim et al. 2010). We isolated AcRMD daughters encoding anti-CD20-IgG under the control of either the p6.9 or the polyhedrin promoters because the latter is activated during the very late phase of infection and is widely used to drive recombinant gene expression in baculoviral vectors, whereas the former is activated somewhat earlier, during the late phase of infection, and can sometimes provide higher efficiencies of protein secretion (Sridhar et al. 1993; Rankl et al. 1994; Bozon et al. 1995; Kost et al. 1997; Toth et al. 2011). We then infected Sf9 cells with these viruses, designated Ac-αCD20-IgG, Acp6.9-αCD20-IgG, AcRMD-αCD20-IgG, or AcRMDp6.9-αCD20-IgG, and measured the relative amounts of anti-CD20-IgG produced and secreted by viruses encoding the heavy and light chains under the control of the two different promoters. The results showed that all four baculovirus vectors induced production and secretion of the anti-CD20-IgG heavy and light chains (FIG. 10). The results also showed that cells infected with the baculovirus vectors encoding anti-CD20-IgG under the control of the late p6.9 promoters secreted higher levels of heavy and light chains than those infected with the baculovirus encoding this product under the control of the very late polyhedrin promoters. Together with the lower levels of heavy and light chains observed in the intracellular fraction, these results indicated that the p6.9-based vectors provided higher efficiencies of anti-CD20-IgG secretion than the polyhedrin-based vectors (FIG. 10). Based on these results, we used Acp6.9-αCD20-IgG and AcRMDp6.9-αCD20-IgG for the remainder of the experiments described in this study.

Acp6.9-αCD20-IgG or AcRMDp6.9-αCD20-IgG were used to infect Sf9 or Tni PRO™ cells and the anti-CD20-IgG secreted by all four virus-cell combinations was affinity purified, resolved by SDS-PAGE under reducing or non-reducing conditions with commercial human IgG as a control, and then stained with Coomassie Brilliant Blue. The results showed that each anti-CD20-IgG preparation analyzed under reducing conditions contained approximately equal proportions of the heavy and light chains, indicating that each virus-cell combination produced and secreted properly assembled forms of anti-CD20-IgG (FIG. 11). This was confirmed by SDS-PAGE analysis under non-reducing conditions, which revealed that each anti-CD20-IgG preparation migrated with relative electrophoretic mobilities that were consistent with their calculated molecular weights (˜165 kDa) and comparable to the human IgG control (FIG. 11). Further analysis showed that the anti-CD20-IgG heavy chain produced by Acp6.9-αCD20-IgG-infected Sf9 cells was PNGase-F-sensitive and AAL-reactive, indicating that it had core α1,6-fucosylated N-glycans (FIG. 12). In contrast, the anti-CD20-IgG heavy chain produced by AcRMDp6.9-αCD20-IgG-infected Sf9 cells was PNGase-F-sensitive, but not AAL-reactive, indicating that it had non-fucosylated N-glycans (FIG. 12). The anti-CD20-IgG heavy chain produced by Acp6.9-αCD20-IgG-infected Tni PRO™ cells was PNGase-F-resistant and AAL-reactive, presumably because its N-glycans were α1,3-fucosylated in these cells (FIG. 12). In contrast, the anti-CD20-IgG heavy chain produced by AcRMDp6.9-αCD20-IgG-infected Tni PRO™ cells was PNGase-F-sensitive and did not react with AAL (FIG. 12). Together, these results strongly suggested that early expression of Rmd by the vectors derived from the AcRMD parent blocked core fucosylation of anti-CD20-IgG in the baculovirus-insect cell system.

To confirm and extend these results using a more direct approach, we enzymatically released, permethylated, and used MALDI-TOF MS to analyze the N-glycans from anti-CD20-IgG preparations purified from various virus-cell combinations, as described in Materials and methods. The N-glycan profiles observed with the anti-CD20-IgG from Acp6.9-αCD20-IgG-infected Sf9 cells included four peaks assigned as mono-fucosylated N-glycans (m/z 1345.7, 1590.8, 1794.9, and 1999.0; FIG. 13A), which are presumably core α1,6-fucosylated, based on the PNGase-F-sensitivity of the heavy chain (FIG. 12). In contrast, none of these mono-fucosylated peaks were detected among the N-glycans isolated from the anti-CD20-IgG produced by AcRMDp6.9-αCD20-IgG-infected Sf9 cells (FIG. 13B). In addition, the loss of the fucosylated N-glycan peaks was accompanied by increased N-glycan peaks corresponding to their non-fucosylated counterparts, especially those with m/z values of 1171.6 and 1416.7 (FIGS. 13A and 13B). The N-glycan profiles observed with the anti-CD20-IgG from Acp6.9-αCD20-IgG-infected Tni PRO™ cells included seven peaks assigned as fucosylated N-glycans, three of which were mono-fucosylated (m/z 1345.7, 1590.8, and 1835.9) and four of which were di-fucosylated (m/z 1315.7, 1519.8, 1764.9, and 2173.1; FIG. 13C). The di-fucosylated N-glycans represented ˜65.5% of total (FIG. 13C), reflecting the high levels of core α1,3-fucosylation observed in Tni PRO™ and High Five™ cells (FIG. 2). In contrast, no fucosylated N-glycans were detected in the profiles obtained with the anti-CD20-IgG produced by AcRMDp6.9-αCD20-IgG-infected Tni PRO™ cells (FIG. 13D) and, again, the loss of fucosylated N-glycan peaks was accompanied by increases in the N-glycan peaks corresponding to their non-fucosylated counterparts (FIGS. 13C and 13D; m/z 1171.6 and 1416.7). It is tempting to speculate that the large increase in the m/z 1416.7 peak, which represents an N-glycan with a single N-acetylglucosamine residue on its non-reducing end, reflects the relative inability of Sf-FDL (Geisler et al., 2008) to remove this sugar from non-fucosylated substrates. Regardless, the mass spectrometric analysis of the N-glycans isolated from anti-CD20-IgG produced by the Rmd-negative or Rmd-positive baculovirus vectors clearly demonstrated that the new vector designed to express Rmd early in infection can block recombinant glycoprotein fucosylation in the baculovirus-insect cell system.

Discussion

Core α1,3-fucosylation generates an immunogenic sugar epitope that has significantly hindered development and utilization of insect-based systems, including the baculovirus-insect cell system for the production of recombinant glycoproteins for therapeutic drug and diagnostic applications in human medicine. In addition, core α1,6-fucosylation of certain types of recombinant antibodies in this system and others represses their effector functions. Thus, the basic purpose of this study was to develop new tools that could be used to produce non-fucosylated recombinant glycoproteins, including antibodies, in insect-based systems, including the baculovirus-insect cell system.

It is well established that High Five™, which is a widely used insect cell line derived from Trichoplusia ni, produces high levels of immunogenic core α1,3-fucosylated N-glycans. In the first part of this study, we showed that Tni PRO™ cells, also derived from Trichoplusia ni, produce high levels of immunogenic core α1,3-fucosylated N-glycans, as well. This finding is relevant because High Five™ and Tni PRO™ cells can potentially produce recombinant glycoproteins at higher levels than other insect cell lines (Davis et al. 1992; Krammer et al. 2010). Tni PRO™ cells have the additional advantage of being easier to culture in suspension and, unlike High Five™ cells (Dee et al. 1997; Taticek et al. 1997; Savary et al. 1999), are directly transferrable without adaptation from serum containing to serum-free ESF 921 medium (unpublished observations). In the course of this study, we found that a mouse IgG2a-Fc domain and a therapeutic anti-CD20-IgG were both core α1,6-fucosylated when produced in Sf9 cells and core α1,6- and core α1,3-fucosylated when produced in High Five™ or Tni PRO™ cells. While these results were not surprising in view of previous literature, they were important because they clearly justified an effort to block recombinant glycoprotein fucosylation in the baculovirus-insect cell system.

To accomplish this goal, we focused on a bacterial enzyme, Rmd, which consumes the direct precursor to GDP-L-fucose and was expected to block recombinant glycoprotein fucosylation in insect cell lines. In fact, previous work had shown that core α1,6-fucosylation could be blocked in CHO cells genetically transformed to overexpress this enzyme (von Horsten et al. 2010). Thus, our analogous initial approach was to transform Sf9 and High Five™ cells to constitutively express Rmd under the control of the AcMNPV ie1 promoter. We successfully isolated Sf9 and High Five™ cell subclones that initially had fucosylation-negative phenotypes and were able to produce a non-fucosylated recombinant glycoprotein. However, this phenotype was unstable, as both insect cell lines reverted to fucosylation-positive phenotypes after a relatively small number of passages in culture. This completely surprising result revealed that the cell engineering approach previously used to block core α1,6-fucosylation in CHO cells cannot be successfully applied to block core α1,6- and/or α1,3-fucosylation in insect cell systems. Thus, we sought to develop a new approach that involved glycoengineering the baculovirus vector, rather than the host.

We assessed the efficacy of our proposed vector engineering approach by co-infecting Sf9 and Tni PRO™ cells with separate baculovirus vectors encoding Rmd under the control of the ie1 promoter or mIgG2a-Fc under the control of the polyhedrin promoter. Analysis of the resulting N-glycosylation patterns showed that early expression of Rmd could block core fucosylation of mIgG2a-Fc produced at a later time of infection. This encouraged us to create a novel baculovirus vector designed not only to express Rmd immediately after infection, but also to enable quick and efficient isolation of daughter vectors capable of expressing any recombinant glycoprotein of interest at later times after infection. After isolating and characterizing AcRMD, the parent baculovirus vector, we used it to isolate a daughter encoding the heavy and light chains of rituximab, an anti-CD20-IgG, under the control of dual, back-to-back p6.9 promoters. We chose the late p6.9, rather than the very late polyhedrin promoter to drive expression of this biotechnologically relevant recombinant glycoprotein because we found that it provided a higher efficiency of IgG secretion. Finally, we showed that this novel baculovirus vector could be used to produce recombinant anti-CD20-IgG with no detectable core α1,6- or α1,3-fucosylation. This conclusion was based on results obtained from several different methods of N-glycan analysis, including endoglycosidase treatments, lectin blotting assays, and MALDI-TOF MS profiling. It is important to note that our MALDI-TOF MS profiling results indicating there were no detectable fucosylated N-glycans on the recombinant anti-CD20-IgG produced by the Rmd-positive baculovirus vector were obtained using N-glycans isolated with a highly active form of PNGase-A (PNGaseAr; New England Biolabs), which effectively removes core α1,3-fucosylated structures, and with glycan detection levels in the picomolar range. It is also important to note that our MALDI-TOF MS results revealed no detectable N-glycans containing any deoxyhexose, indicating that GDP-rhamnose is a dead-end product that cannot be utilized for N-glycan modification in the baculovirus-insect cell system.

There are clear advantages to engineering the baculovirus vector, rather than host cell lines to block recombinant glycoprotein fucosylation in baculovirus-insect cell systems. One is that any investigator familiar with these systems can use the new AcRMD vector in conjunction with an established linearized viral DNA (Kitts and Possee 1993) approach for homologous recombination with familiar, even pre-existing baculovirus transfer plasmids to efficiently isolate daughter baculovirus vectors encoding their favorite recombinant glycoprotein. Another is that the resulting daughter vectors can be used to produce non-fucosylated forms of that product in standard, familiar, commercially available insect cell lines, such as Sf9, Sf21, High Five™, Tni PRO™, Ea4, S2, or S2R+, even if the investigators favorite cell line normally produces high levels of α1,3-fucosylated N-glycans. This eliminates the need to maintain specialized cell lines transformed to block recombinant glycoprotein fucosylation, which might require different growth media and/or conditions and have different growth properties, all of which complicate routine cell culture operations. Overall, it is highly advantageous to be able to produce non-fucosylated recombinant glycoproteins by simply replacing the recombinant baculovirus used for standardized production runs with its AcRMD counterpart, without having to alter or re-optimize existing protocols. Most importantly, engineering the virus eliminates the problem of genetic instability associated with engineering the insect cell lines, because low passage virus stocks can be produced, checked, and stored in a biologically inert state. In contrast, cell lines transformed to constitutively express Rmd must be maintained in culture and, therefore, subjected to constant selective pressure, which can drive loss of the fucosylation-negative phenotype, as observed in this study. Mammalian cell expression systems engineered to produce non-fucosylated recombinant glycoproteins, such as FG1, CHO SM 3G1, CHO FUT8^(−/−), RMD-CHO, and CHO-DUKX (Imai-Nishiya et al. 2007; Kanda et al. 2007; Malphettes et al. 2010; von Horsten et al. 2010; Zhong et al. 2012), are also subject to the potential problem of long-term instability. In addition, mammalian cells have a salvage pathway that can produce GDP-L-fucose using exogenous fucose, which is a common contaminant of many cell culture components. This salvage pathway can rescue recombinant glycoprotein fucosylation in all mammalian cell lines engineered to eliminate this modification except the FUT8 knockout line. This is unlikely to be a problem in the baculovirus-insect cell system because insects do not appear to encode the enzymes involved in the salvage pathways for GDP-L-fucose biosynthesis.

Arguably, the most important feature of the novel baculovirus vector described in this study is its ability to eliminate the immunogenic sugar epitope resulting from core α1,3-fucosylation of recombinant glycoproteins. This will enable investigators to exploit the potentially higher productivity of insect cell lines derived from Trichoplusia ni for recombinant glycoprotein manufacturing (Davis et al. 1992; Krammer et al. 2010). It will also expand the utility of the baculovirus-insect cell system to include production of recombinant glycoproteins for human clinical applications, including therapeutics and diagnostics. Historically, the production of recombinant glycoproteins for therapeutic use in humans has not been a legitimate application of the baculovirus-insect cell system. This study shifts this paradigm because the new baculovirus vector described herein can block core α1,3-fucosylation in insect cell lines glycoengineered to produce humanized, terminally sialylated N-glycans. The combination of these emerging tools constitutes a novel baculovirus-insect cell platform that can be used to manufacture safe and efficacious glycoproteins for human therapy.

Another important feature of the novel baculovirus vector described in this study is its ability to block core α1,6-fucosylation, which is a common modification of a conserved N-glycan on the Fc domain that represses the effector functions of certain types of therapeutic antibodies. As noted above, core α1,6-fucosylation has been blocked in other expression systems in efforts to produce therapeutic antibodies with enhanced effector functions and, therefore, higher efficacy at lower doses (reviewed by Yamane-Ohnuki and Satoh 2009). Sf9 and Tni PRO™ cells infected with the AcRMD daughter vector encoding anti-CD20-IgG produced a non-fucosylated form of this antibody, which is expected to have enhanced effector function, based on a significant body of previous literature (Shields et al. 2002; Shinkawa et al. 2003; Li et al. 2013; reviewed by Lim et al. 2010; Owen and Stewart 2012). Moreover, while many recombinant antibodies have been produced in the baculovirus-insect cell system (reviewed by Cérutti and Golay 2012), most were expressed using dual, back-to-back, very late polyhedrin and p10 promoters to express the heavy and light chains and the heavy chain product. In this study, we found that expression of the heavy and light chains under the control of dual, back-to-back, late p6.9 promoters separated by the second intron from the Drosophila white gene provided a higher secretion efficiency than the analogous arrangement of very late polyhedrin promoters (FIG. 10). This result is consistent with those obtained in similar studies on the relationship between the timing of promoter activation and the efficiency of recombinant glycoprotein processing (Sridhar et al. 1993; Rankl et al. 1994; Bozon et al. 1995; Kost et al. 1997; Toth et al. 2011).

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope of the present invention, as set forth in the following claims. 

What is claimed is:
 1. A method for production of a non-fucosylated protein in insect larvae, comprising a) providing insect larvae; b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an expression control sequence active later in infection, thereby producing non-fucosylated proteins, wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector; c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose; and d) isolating said protein of interest, wherein said RMD encoding nucleic acid is the reverse complement of nucleotides 10034-10948 of SEQ ID NO:
 1. 2. The method of claim 1, wherein said nucleic acid molecule encoding the enzyme RMD is operably driven by an immediate early expression control sequence for expression immediately after infection.
 3. The method of claim 2, wherein said immediate early expression control sequence comprises the ie1 promoter.
 4. The method of claim 3, wherein said expression control sequence active later in infection comprises the p6.9 promoter.
 5. A method for producing at least one protein of interest lacking fucose, comprising a) providing insect cells; b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an expression control sequence active later in infection, thereby producing non-fucosylated proteins, wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector; c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose; and d) isolating said at least one protein of interest, wherein said RMD encoding nucleic acid is the reverse complement of nucleotides 10034-10948 of SEQ ID NO:
 1. 6. The method of claim 5, wherein said RMD enzyme and said protein of interest are encoded by a single recombinant baculoviral vector and expressed sequentially at earlier and later times of infection.
 7. The method of claim 5, wherein said nucleic acid encoding said enzyme and said protein of interest are on separate baculovirus vectors and expressed sequentially at earlier and later times of infection.
 8. The method of claim 5, wherein said protein of interest is a therapeutic protein.
 9. The method of claim 5, wherein said baculovirus vector comprises SEQ ID NO:
 1. 10. The method of claim 5, wherein said baculovirus vector comprises SEQ ID NO: 1, wherein nucleotides 845-1564 are deleted.
 11. The method of claim 5, wherein said expression control sequence active later in infection comprises the p6.9 promoter.
 12. The method of claim 5, wherein said immediate early control sequence comprises a promoter selected from the group consisting of ie1, ie2, ie0, etl, and gp64, insect actin, tubulin, a ubiquitin promoter; RSV promoter, copia, gypsy promoter and a cytomegalovirus IE promoter.
 13. The method of claim 5, wherein said inducible promoter is selected from the group consisting of baculovirus delayed early, late, and very late promoters, an hsp70 promoter, a metallothionein promoter and a tetracycline-regulated promoter.
 14. The method of claim 5, wherein said expression control sequence active later in infection comprises a promoter selected from the group consisting of a promoter from baculovirus delayed early, late, and very late promoters.
 15. The method of claim 5, wherein said immediate early expression control sequence comprises a promoter and an enhancer element that increases activity of said promoter.
 16. The method of claim 15, wherein said enhancer is hr5.
 17. The method of claim 5, wherein said protein of interest is selected from the group consisting of an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, and a blood clotting factor.
 18. The method of claim 5, wherein said insect cells are selected from the group consisting of Sf9, Sf21, Tn368, BTI-Tn-5Bl-4, Ea4, Ao38, BmN, S2, and S2R+.
 19. The method of claim 5, wherein said nucleic acid molecule encoding the enzyme RMD is operably driven by an immediate early expression control sequence for expression immediately after infection.
 20. The method of claim 19, wherein said immediate early expression control sequence comprises the ie1 promoter.
 21. The method of claim 20, wherein said expression control sequence active later in infection comprises the p6.9 promoter. 